1. Field of the Invention
The present invention relates to the field of nanomaterials and specifically to nanopore devices and sensors. In particular, the invention relates to sensing and manipulating and sensing ions and carbohydrates using ionic current measurements on a nanoscale.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Nanopore Ionic Current Modulators
Solid state nanopores are of great interest as stable structures that can be used to mimic biological channels, for the size-selective synthesis of nanoparticles or as nanoscale sensors. Conical, or asymmetric, nanopores are a distinct category of nanochannels that display voltage-gated ion current and can behave as nanofluidic diodes, i.e. they exhibit ionic current rectification. Several groups have developed electrical sensors utilizing ion current measurements across membranes containing asymmetric nanopores (Harrell, C. C. et al., Resistive-pulse DNA detection with a conical nanopore sensor. Langmuir 22, 10837-10843, doi:10.1021/1a061234k (2006); Kececi, K., et al. Resistive-pulse detection of short dsDNAs using a chemically functionalized conical nanopore sensor. Nanomedicine 3, 787-796, doi: 10.2217/17435889.3.6.787 (2008); Sexton, et al., Developing synthetic conical nanopores for biosensing applications. Mol. Bia Syst. 3, 667-685, doi:10.1039/b708725j (2007); Au, M., et al. Biosensing with Functionalized Single Asymmetric Polymer Nanochannels, Macromol. BioscL 10, 28-32, doi:10.1002/mabL200900198 (2010)). Such devices are generally prepared by a track-etching method. Quartz based nanopores, fabricated from quartz capillaries, exhibit many of the same electrical properties but are rapidly prepared using a laser puller. Quartz conical nanopores, also called nanopipettes, exhibit many properties of other asymmetric nanochannels and are advantageous in that the pore can be manipulated with high spatial resolution, a property that has been used to image cells at the nanoscale.
Investigations with conical nanopores have given rise to new chemical and electrical phenomena that challenge existing ideas about bulk materials. Recently, ion current oscillations were observed with rectifying conical nanopores (2 to 8 nm diameter) in polyethylene terepthalate (PET) films, and were attributed to dynamic precipitation in the pore caused by voltage-induced concentration of weakly soluble salts. Current oscillations in much larger pores of silicon nitride or borosilicate glass can be generated at the interface of two solvents using organic molecules with differential solubility. These phenomena offer a new way to electrically monitor nonequilibrium events such as precipitation in real time and at the nanoscale.
Nanopore Sensors
The stability and ability to mimic biological channels make nanopore-based platforms candidates for studying (bio) molecular interactions. Solid-state nanopores are stable, their diameter can be controlled through the fabrication process and they can be integrated into devices and arrays. Furthermore, their surface properties can be easily tuned by chemical functionalization, allowing the development of chemical and biochemical responsive nanopores. Nanopore-based sensors have incorporated receptors including proteins, DNA, aptamers, ligands, and small biomolecules, allowing a variety of analytes to be targeted. Essential to the sensitivity of many solid-state nanopore sensors is the property of ion current rectification (ICR), arising from the selective interaction between ions in solution and the surface of a charged, asymmetrically shaped nanochannel, or conical nanopore. Nanomaterials exhibiting ICR and used as sensors include track-etched nanopores in polymer membranes and quartz nanopipettes. In either case, a key challenge is the surface modification with appropriate receptors.
Conical quartz nanopores have also been functionalized for sensing applications [See, for example, Sa, N., Fu, V. & Baker, I. A. “Reversible Cobalt Ion Binding to Imidazole-Modifled Nanopipettes.” Anal Chem., 82, 9663-9666, doi:10.1021/ac102619j (2010); Fu, Y., Tokuhisa, H. & Baker, I. A. “Nanopore DNA sensors based on dendrimer-modified nanopipettes.” Chem Commun (Comb), 4877-4879, doi:10.1039Jb910511e (2009); Umehara, S., Karhanek, M., Davis, R. W. & Pourmand, N. “Label-free biosensing with functionalized nanopipette probes.” Proceedings of the National Academy of Sciences 106, 4611-4616, doi:10.1073/pnas.0900306106 (2009); Actis, P., Mak, A. & Pourmand, N. “Functionalized nanopipettes: toward label-free, single cell biosensors.” Bioanalytical Reviews 1, 177-185, doi:10.1007/s12566-010-0013-y (2010); Actis, P., Jejelowo, 0. & Pourmand, N. “Ultrasensitive mycotoxin detection by STING sensors.” Biosensors and Bioelectronics 26, 333-337 (2010)].
To date, the reversible binding of analytes with nanopore sensors has proven challenging. However, this is a critical issue if such devices are to be used for applications such as continuous monitoring or repeated measurements with one sensor. Multiple uses for a single sensor will also overcome problems in reproducibly producing pores of the same size, which limits quantitative measurements for many sensors reported in the literature. For such applications, the nanopipette is a promising platform as the sensor tip can be precisely and rapidly manipulated between samples, or within a single sample, with nanoscale precision. To date, functionalized nanopores responsive to pH have shown the best properties in terms of rapidly reversible and selective behavior. Nanopipettes functionalized with imidazole and responding to cobalt ions can be regenerated by immersion in solution of low pH, reprotonating the ligand (Sa, N.; Fu, V.; Baker, L. A., Reversible Cobalt Ion Binding to Imidazole-Modified Nanopipettes. Anal. Chem. 2010, 82 (24), 9963-9966).
Transport through nanopores can be modified by a variety of external stimuli including voltage and pressure (see Lan, W.-J.; Holden, D. A.; White, H. S., Pressure-Dependent Ion Current Rectification in Conical-Shaped Glass Nanopores. J. Am. Chem. Soc. 2011, 133 (34), 13300-13303.). Simply changing the salt gradient across a nanopore can affect transport, and this effect was used to focus DNA for resistive-pulse measurements (see Wanunu, M.; Morrison, W.; Rabin, Y.; Grosberg, A. Y.; Meller, A., Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient. Nat Nano 2010, 5 (2), 160-165.). Nanopores can also be engineered to respond to stimuli such as solvent polarity. This can be achieved with so-called “hairy nanopores,” in which the nanopore is decorated with polymers (see Peleg, O.; Tagliazucchi, M.; Kröger, M.; Rabin, Y.; Szleifer, I., Morphology Control of Hairy Nanopores. ACS Nano 2011, 5 (6), 4737-4747.). Several artificial nanopores have been engineered for pH-sensitivity using surface modification. Conical nanopores have been modified with receptors for binding other charged species, which likewise modulate current rectification. Targets have included nucleic acids, metal ions, proteins, and small molecules. In the cases of large biomolecules, such as nucleic acids and proteins, physical blocking of the pore likely plays a role in addition to modulation of the surface charge. To date, the modulation of current rectification with small, uncharged species has proved difficult. However, such a system would expand the stimuli for responsive nanopores to include drugs, peptides, and carbohydrates.
Glucose/Diol Sensing
Carbohydrate recognition is essential to monitoring of blood glucose (Kondepati, et al. Anal. Bioanal. Chem. 388, 545-563 (2007). Detection and quantification of carbohydrates can also be used in bioprocess monitoring and for medical diagnostics based on metabolic saccharides, nucleotides, or glycoproteins (Timmer, et al. Curr. Op. Chem. Biol. 11, 59-65 (2007). Most electrochemical methods for measuring glucose rely on redox enzymes such as glucose oxidase (Oliver, et al. Diabetic Med. 26, 197-210 (2009). The most common artificial receptors use boronic acids, which have predominately been used for optical probes (Mader & Wolfbeis, Microchimica Acta 162, 1-34 (2008). Non-enzymatic methods for electrochemical measurement of glucose have also been developed, mostly relying on oxidation of glucose (Park, et al. Anal. Chim. Acta 556, 46-57 (2006); E. T Chen, Nanopore structured electrochemical biosensors, US 2008/0237063).
To date, there has been very little reported in the literature on nanofluidic pores that respond to carbohydrates. Nanopore analytics have been used to detect small molecules using resistive-pulse methods, but the technique is generally more suited to proteins and other macromolecules. Oligosaccharides on the order of MW 500 to 10,000 have been discriminated using resistive-pulse techniques with alpha-hemolysin pores.
One example of receptor-modified nanopores uses with a covalently attached HRP enzyme, which is then conjugated supramolecularly to Con A, a saccharide-binding protein which interacts with mannose units on the HRP molecule (Ali, et al. Nanoscale 3, 1894-1903 (2011). Addition of monosaccharides (galactose and glucose) competes with the Con A, changing the ion current through the pore. Two recent examples make use of boronic acid as a chemical receptor, where the receptor is attached covalently to the walls of artificial nanopores (Sun, Z.; Han, C.; Wen, L.; Tian, D.; Li, H.; Jiang, L., pH gated glucose responsive biomimetic single nanochannels. Chem. Commun. (Cambridge, U. K.) 2012.; Nguyen, Q. H.; Ali, M.; Neumann, R.; Ensinger, W., Saccharide/glycoprotein recognition inside synthetic ion channels modified with boronic acid. Sensors and Actuators B: Chemical 2012, 162 (1), 216-222.). In the case of the former, an acidic solution was required to reverse the saccharide binding and restore the signal. In the latter, reversible binding was not demonstrated.
Despite many recent advances in nanopore fabrication and surface chemistry, the work cited above shows that there is a need for new schemes to modulate ion current using carbohydrates as an external stimulus. This problem may be addressed with new functional materials that can interface with nanopores.
Specific Patents and Publications
Karhanek et al. in US Patent Application Publication 2010/0072080, published on Mar. 25, 2010, disclose methods and devices comprising a nanopipette having thereon peptide ligands for biomolecular detection, including of peptides and proteins.
Siwy et al. in U.S. Pat. No. 7,708,871, issued on May 4, 2010, disclose an apparatus having a nanodevice for controlling the flow of charged particles in an electrolyte. Such apparatus comprises an electrolytic bath container divided by a polymeric membrane foil for controlling the flow of charged particles in an electrolyte.
Sa et al. in Analytical Chemistry 2010, 82 (24), pp 9963-9966 disclose that quartz nanopipettes modified with an imidazole-terminated silane respond to metal ions (Co2+) in solution. The response of nanopipettes was evaluated through examination of the ion current rectification ratio. When nanopipettes were cycled between solutions of different pH, adsorbed Co2+ was released from the nanopipette surface, to regenerate binding sites of the nanopipette.
Umehara et al. in Proceedings of the National Academy of Sciences, vol 106, pages 4611-4616, Mar. 24, 2009, disclose a label-free, real-time protein assay using functionalized nanopipette electrodes. Electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface were shown to affect ionic current flowing through a 50-nm pore.
Umehara et al. “Current Rectification with Poly-L-lysine Coated Quartz nanopipettes,” Nano Lett. 6(11):2486-2492 (2006) discloses current responses of noncoated and Poly-1-lysine coated nanopipettes using a nanopipette in a bath solution.
Karhanek M., Kemp J. T., Pourmand N., Davis R. W. and Webb C. D, “Single DNA molecule detection using nanopipettes and nanoparticles,” Nano Lett. 2005 February; 5(2):403-7 discloses that single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The disclosed set up uses a voltage clamp circuit, which utilized a single detecting electrode in a bath to detect nanoparticle-DNA current block.
Ying, Liming in Biochemical Society Transactions, vol 37, pages 702-706, 2009, reviews nanopipettes and their use in nanosensing and nanomanipulation of ions, molecules (including biomolecules) and cells.
Borghs, Gustaaf, et al. in WO 2006/000064, published on Jan. 5, 2006, disclose a nanofluidic device for controlling the flow of charged carriers through a nanopore extending through a membrane.
Sunkara, et al. in US Patent Application Publication 2005/0260119, published on Nov. 24, 2005, disclose a method of synthesizing tubular carbon nanostructures in the form of tapered whiskers, termed nanopipettes, using microwave plasma assisted chemical vapor deposition method.
Chen US 20080237063 published Oct. 2, 2008, entitled “Nanopore structured electrochemical biosensors,” discloses a biosensor having a nanopore structured and catalytically active cyclodextrin attached thereto for direct measurement of glucose.
Choi et al., “Biosensing with conically shaped nanopores and nanotubes,” Phys. Chem. Chem. Phys. 8:4976-4988 (2006) discusses the preparation and characterization of conical nanopores synthesized using a track-etch process. The design and function of conical nanopores that can rectify the ionic current that flows through these pores under an applied transmembrane potential is also disclosed.
Li et al., “Development of boronic acid grafted random copolymer sensing fluid for continuous glucose monitoring,” Biomacromolecules 10(1):113-118 (2009) discloses biocompatible copolymers poly(acrylamide-ran-3-acrylamidophenylboronic acid) (PAA-ran-PAAPBA) for viscosity based glucose sensing.
Sun, Z.; Han, C.; Wen, L.; Tian, D.; Li, H.; Jiang, L., pH gated glucose responsive biomimetic single nanochannels. Chem. Commun. (Cambridge, U. K.)(2012) describe a track-etched conical nanochannel in polyethylene teraphthalate (PET) covalently modified with phenylboronic acid receptors.
Nguyen, Q. H.; Ali, M.; Neumann, R.; Ensinger, W., Saccharide/glycoprotein recognition inside synthetic ion channels modified with boronic acid. Sensors and Actuators B: Chemical 2012, 162 (1), 216-222.) describe a track-etched conical nanochannel in polyethylene teraphthalate (PET) covalently modified with phenylboronic acid receptors. The channel responds to monosaccharides as well as glycoproteins.